Bdellovibrio’s prey-independent lifestyle is fueled by amino acids as a carbon source

Abstract Identifying the nutritional requirements and growth conditions of microorganisms is crucial for determining their applicability in industry and understanding their role in clinical ecology. Predatory bacteria such as Bdellovibrio bacteriovorus have emerged as promising tools for combating infections by human bacterial pathogens due to their natural killing features. Bdellovibrio’s lifecycle occurs inside prey cells, using the cytoplasm as a source of nutrients and energy. However, this lifecycle supposes a challenge when determining the specific uptake of metabolites from the prey to complete the growth inside cells, a process that has not been completely elucidated. Here, following a model-based approach, we illuminate the ability of B. bacteriovorus to replicate DNA, increase biomass, and generate adenosine triphosphate (ATP) in an amino acid-based rich media in the absence of prey, keeping intact its predatory capacity. In this culture, we determined the main carbon sources used and their preference, being glutamate, serine, aspartate, isoleucine, and threonine. This study offers new insights into the role of predatory bacteria in natural environments and establishes the basis for developing new Bdellovibrio applications using appropriate metabolic and physiological methodologies. Key points • Amino acids support axenic lifestyle of Bdellovibrio bacteriovorus. • B. bacteriovorus preserves its predatory ability when growing in the absence of prey. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-024-13250-y.


Introduction
A complete understanding of the physiological and nutritional requirements that enable optimal cell growth plays an important role in the use of microorganisms in biotechnological, industrial, and medical applications.In recent years, there has been an increase in new approaches to efficiently growing microorganisms by taking advantage of new knowledge of their physiology (Chubukov et al. 2014;Monk et al.Cristina Herencias and Virginia Rivero-Buceta are contributed equally and should be considered as both first authors.2014).Bdellovibrio bacteriovorus is a gram-negative predatory bacterium belonging to the group of Bdellovibrio and like organisms (BALOs) that prey on other gram-negative bacteria.This species is ubiquitous, given that it can be found in soil, aquatic environments, and human commensal microbiota (Herencias et al. 2020a).Through a periplasmic predation strategy, Bdellovibrio uses the nutrients in its prey's cytoplasm as carbon and energy sources for growth.Once the prey is exhausted, Bdellovibrio septates into its progeny and finally lyses the prey cell to begin another attack (Fig. 1a).Under certain conditions (limited nutrients/prey), certain Bdellovibrio strains can change their lifestyle strategy and develop as prey-independent cells (Hobley et al. 2012).These Bdellovibrio variants can grow in the absence of prey bacteria, and their genotypic characterisation has revealed that they possess various mutations in genes related to the transduction of type IV pili (T4P) interactions, critical for prey recognition and attachment (Seidler and Starr 1969;Barel and Jurkevitch 2001;Evans et al. 2007;Chang et al. 2011;Morehouse et al. 2011;Roschanski et al. 2011;Capeness et al. 2013;Kaplan et al. 2023).
Derived from its lifestyle, multiple applications of the predators in various fields have been proposed, such as biocontrol agents (food preservation and water treatment), living antibiotics (to combat multiresistant pathogens), and hydrolytic enzyme producers (Herencias et al. 2020a;Lai et al. 2023).
Several biochemical studies have highlighted aspects of the energetic metabolism of Bdellovibrio using diverse substrates such as amino acids and RNA (Hespell et al. 1973;Hespell et al. 1974;Hespell and Odelson 1978;Rosson and Rittenberg 1979;Nelson and Rittenberg 1981;Rosson and Rittenberg 1981).Analysis of the Bdellovibrio genome revealed the presence of complete biosynthetic and degradation pathways for fatty acids; however, the lack of a phosphotransferase system makes the use of carbohydrates unlikely (Rendulic et al. 2004;Herencias et al. 2020b).More recent studies have examined the possibility of growing predator cells in a rich medium, offering new biotechnological perspectives (Sathyamoorthy et al. 2021).Cell survival is minimally altered during prey starvation in the attack phase (Sathyamoorthy et al. 2021), and there is a slight increase in cell size and stimulation of protease secretion (Dwidar et al. 2017).
Currently, the precise nutritional requirements that allow for the growth of these predators are not entirely elucidated, mainly due to this organism's host-dependent nature (Lai et al. 2023).Analysing which metabolites from the prey cells are taken up during intraperiplasmic growth is a challenge, given the vast repertoire of molecules contained in the prey cell.
We have reported a genome-scale metabolic model of this predatory bacterium (iCH457) to characterise and predict the metabolic potential and behavior of the biphasic  (Herencias et al. 2020b).Our model simulations have consistently postulated the growth capacity of Bdellovibrio in a rich amino acid medium in the absence of prey, based on its metabolic competencies.
The present study aimed to determine the metabolic capabilities of the host-dependent B. bacteriovorus 109J, a laboratory reference strain, in a nutrient-rich but preyfree medium, and the uptake of metabolites during cellular growth.A better understanding of how these bacteria grow and their physiological and metabolic requirements will provide insight into their role in various microbial environments and increase the possibility of producing and applying predators rationally and safely in industry and clinical practice.

In silico growth simulations
Biomass production and amino acids consumption were calculated using the dynamic Flux Balance Analysis (dFBA) of the COBRA toolbox 3.0 (Heirendt et al. 2018) in the environment MATLAB R2022a (MathWorks, Natick, MA, USA) using the Gurobi Solver (v9.5.2,Gurobi Inc.).Biomass was set as the objective function and the bounds of the exchange fluxes were set as the rich in silico medium as described previously (Herencias et al. 2020b).Amino acids exchange reactions were set as "substrateRxns", and "initConcetrations" of amino acid or dipeptides were based on Lysogeny Broth (Sezonov et al. 2007).Since L-alanine is supplied in the form of dipeptides, the initial concentration of L-alanine was set in 0.001 mM, as well as the other unsupplied dipeptides, to avoid being set unlimited for the function dFBA.Amino acids and dipeptides transporters are included in the metabolic model, based on previous studies (Herencias et al. 2020b).(Herencias et al. 2017;Saralegui et al. 2022).To remove the prey, the co-cultures were filtered twice through a 0.45 µm filter (Sartorius).The axenic cultivation of Bdellovibrio was carried out in PYE10 (10 g L -1 peptone and 10 g L -1 yeast extract) medium.CAV defined medium is composed of 200 µM of each amino acid: phenylalanine, glutamate, aspartate, threonine, serine, glycine, proline, isoleucine, leucine, valine and alanine.CAV medium was supplemented with a solution of trace elements (composition 1000 × 2.78 FeSO 4

B. bacteriovorus viability calculation
Predator viability was counted as plaque forming units per milliliter (pfu mL −1 ).It was calculated from a culture performing serial dilution from 10 -1 to 10 -7 in Hepes buffer and developing on the lawn of prey after 48-72 h of incubation at 30 °C by using the double layer method as described previously (Herencias et al. 2017;Saralegui et al. 2022).Briefly, 0.1 mL of the appropriate culture dilution was mixed with an additional 0.5 mL of prey cell suspension of P. putida KT2440 (previously pre-grown in NB and prepared in Hepes buffer OD 600 10) and 4 mL of DNB 0.7 % agar.The final mix was vortexed and plated on DNB solid medium (1.5 % agar).

Biomass calculations
Cellular biomass, expressed in grams of cell dry weight (g CDW) per liter, was determined gravimetrically as previously reported (Martínez et al. 2013).Briefly, 10 ml of culture medium was centrifuged for 15 min at 13000 × g at 4 °C.Cell pellets and the supernatants were separated, subsequently dried at -80 ºC for 24 h, lyophilized, and finally weighed.The biomass calculation was obtained from the pellet weight.

Measurements of ATP intracellular levels
Intracellular ATP levels were determined by an ATP bioluminescence assay kit (ATP Biomass Kit HS, BioThema) according to the manufacturer's instructions.To measure the intracellular ATP, 1 mL of B. bacteriovorus cells was centrifuged for 15 min at 13,000 × g and 4 °C and the pellet was suspended in 1 mL of saline solution (0.85 % NaCl).For each condition, four different experiments were carried out and two technical replicates were measured.To normalize the ATP intracellular values, the number of viable predator cells was also measured by the double-layer method.

Quantification of B. bacteriovorus genome number
The abundance of the predator (number of genomes) was estimated by quantitative PCR (qPCR).A DNA fragment of 121 bp located in the coding region of the housekeeping gene bd2400 (Dori-Bachash et al. 2008) was amplified using the oligonucleotides Bd2400-1 (5´-GCG ACT CCA GAA CAG CAG ATT) and Bd2400-2 (5´-GAA TCC GCG GAC TGC ATT GTA) (Dori-Bachash et al. 2008).qPCR was performed using the SYBR Green (LightCycler® 480 SYBR Green I Master) technology in a LightCycler 480 Real-Time PCR system.Samples were directly analyzed from the culture (containing either prey and predator or predator alone), without DNA extraction.For the calibration curve, genomic DNA was purified from a filtered co-culture of B. bacteriovorus 109J using the IllustraTM bacteria genomicPrep Mini Spin Kit (GE Healthcare) following the instructions of the manufacturers.For the measurements, 200 µL of each sample was collected and stored at -20 °C until analysis.Samples were initially denatured by heating at 95 °C for 5 min, followed by 45 cycles of amplification (95 °C, 10 s; test annealing temperature, 60 °C, 10 s; elongation and signal acquisition, 72 °C, 10 s).For quantification of the fluorescence values, a calibration curve was made using serial dilution from 5 to 5•10 -7 ng of B. bacteriovorus 109J genomic DNA sample.qPCR was performed with triplicate samples from three independent biological experiments.The negative control was achieved with genomic DNA from P. putida KT2440 as a template.The results were analyzed using the 2 −ΔΔCt method and the genome number per mL can be calculated as follows (Livak and Schmittgen 2001;Van Essche et al. 2009): n° of genomes of Bd = (ng of DNA in the sample by qPCR)/(weight of the amplicon), being the weight of the amplicon of 1.24•10 -10 ng.The calculations of concentration (genome mL −1 ) consider that the genome of the predator contains one copy of the bd2400 gene.Genes covering the whole genome were selected to validate the qPCR method and are listed in Table S2 and Table S3.

Correlation between viable cells and genome number
To correlate qPCR data with viable Bdellovibrio cells, we prepared co-cultures with the predator and P. putida KT2440 as prey at different predator-to-prey ratios (1:0.1, 1:1, and 1:10) and initial prey cell numbers (3 × 10⁸ and 6 × 10⁸).A total of 30 co-cultures were incubated for 24 h at 30 °C with shaking at 250 rpm.Plaque-forming units and prey genome numbers were measured at the beginning and end of the predation assay.The data were then transformed to logarithm as previously described (Ogundero et al. 2022) and used to obtain the following linear regression equation: y = 0.48 + 1.2 x as shown in Fig. S1, where "y" represents the measured genome number using qPCR assay and "x" is the calculated viable predator cells.

Amplification of DNA and hit locus sequencing
PCR amplifications were performed in the buffer recommended by the manufacturer adding 0.05 μg of template DNA, 1 U of Phusion DNA polymerase and 0.4 μg of each deoxynucleotide triphosphate.Conditions for amplification were chosen according to the GC content of the oligonucleotides used, provided by the manufacturer (Sigma-Aldrich).DNA fragments were purified by standard procedures using the Gene Clean Turbo Kit (MP Biomedicals).PCR products were purified using the High Pure Purification Kit (Roche Applied Science).The oligonucleotides used to amplify and sequence the different hit-related genes are listed in Table S4.

Whole genomic sequencing
Genomic DNAs of B. bacteriovorus 109J incubated under axenic conditions in HEPES buffer and PYE10 after 24 h were extracted with a PureLink™ Genomic DNA Mini Kit (Invitrogen from Thermo Fisher Scientific).For whole genomic sequencing, the final concentration was adjusted to 50 ng/μL using a NanoPhotometer® bioNova spectrometer (Implen).Genome sequencing was carried out by Plasmidsaurus using Oxford Nanopore technology.Whole Genome Shotgun BioProject has been deposited at DDBJ/ ENA/GenBank under the ID PRJNA1104159.The Sequence Read Archive (SRA) data was submitted under accessions SAMN41069971 and SAMN41069972 for HEPES and PYE10 conditions, respectively.Mapping and single nucleotide polymorphisms (SNP) calling were performed by Geneious Prime version 2020.0.4 created by Biomatters (https:// www.genei ous.com) using B. bacteriovorus 109J (NZ_CP007656) as the reference genome.

Analysis of extracellular metabolites by untargeted GC-TOF-MS
The supernatants of the cultures were collected, frozen at -80 °C and lyophilized.All GC identifications were based on retention times and/or comparisons with commercially available standards.Moreover, the National Institute of Standards and Technology (NIST) (Neto et al. 2016) Mass Spectra Library based on a pre-defined matching criterium (similarity index ≥ 70 % (Scheubert et al. 2017) against mass spectral libraries was used.
Before analyses, samples were derivatized to increase the volatility of polar metabolites.For this purpose, 5-10 mg of the lyophilized supernatant, 300 µL of pyridine and 200 µL of BSTFA (with 1 % TMCS) were added to each sample and heated with stirring at 70 °C for 1 h.Subsequently, the reaction mixture was transferred to the GC autosampler vials for further GC-TOF-MS analyses (Chang and Ho 2014).
Agilent 7890A Gas Chromatography (Agilent Technologies) coupled to Waters Micromass GCT Premier Mass Spectrometer (coupled to COMBI-PAL-GC (EI)) (Waters Corporation, Milford) was used for separation and detection in the GC-TOF-MS setup.A GC column (ZB-5MSplus, Phenomenex) of 30.0 m × 250 µm × 0.25 µm was used.Helium was used as the carrier gas at a flow rate of 1.0 mL min −1 .The split ratio for the injector was set to 1∶10, with a total injection volume of 2 µL.Front inlet and ion source temperatures were both kept at 270 °C.Oven temperature was set to equilibrate at 60 °C for 1 min, before initiation of sample injection and GC run.After sample injection, the oven temperature was increased at a rate of 6 °C min −1 to 325 °C and held at 325 °C for 3 min.The MS detection was operated in EI mode (70 eV) with a detector voltage of 1900 V. Full scan mode with a mass range of m/z 50-800 was used as the data acquisition method.The metabolite identities will be confirmed by targeted analysis.

Phase-contrast microscopy
To monitor the physiology of Bdellovibrio cells, cultures were routinely visualized using a 100× phase-contrast objective and images taken with a Leica DFC345 FX camera.

Statistical analysis
Data sets were analyzed using Prism 6 software (Graph-Pad Software Inc.).Comparisons between two groups were made using Mann-Whitney-test followed by a no-parametric Krustal-Wallis analysis.The relation between the microbial counting and the genome number was analyzed by Pearson correlation.Data was represented using a R custom script and the "ggplot2" package.

Rich medium supports the growth of B. bacteriovorus 109J in the absence of prey
The rational application of predatory bacteria for biotechnological or clinical purposes depends, to a large extent, on the level of understanding of the bacteria's lifecycle and growth requirements.The biphasic lifecycle of B. bacteriovorus can complicate its cultivation at a large scale and its consequent applications; however, recent evidence of its metabolic capabilities for axenic lifestyle will facilitate these processes (Herencias et al. 2020b).To provide further insights on this topic, we employed the curated genome-scale model for B. bacteriovorus (iCH457) to predict cell growth by conducting a dynamic flux balance analysis (dFBA).Combining this approach with the optimization of biomass production under rich medium conditions (rich in silico medium (Herencias et al. 2020b)) it was predicted a specific growth rate of 0.486 h −1 (Fig. 1b).Furthermore, we found an interesting hierarchy in the amino acid consumption being glutamate the first carbon source used, followed by glutamine, serine, threonine, and asparagine (Fig. 1b).
The above in silico results strongly argue in favour of the full capacity of Bdellovibrio to grow axenically using amino acids as carbon and energy sources.To test this hypothesis, we cultivated the bacteria for 96 h in an amino acid-based media containing peptone and yeast extract (PYE10) and monitored growth parameters such as the viable cell count, the number of genomes, the intracellular ATP levels, and the biomass production.The results showed that while the number of viable predators decreased by 1.32 log 10 after 96 h (median log of viable cell number in PYE10 at 0 and 96 h is 9.28 ± 0.25 and 7.97 ± 0.25, respectively), the genome number increased by 0.5 log 10 (median log of genome number in PYE10 at 0 and 96 h is 9.23 ± 0.08 and 9.72 ± 0.23, respectively), suggesting that a number of the surviving cells replicated their DNA without compromised viability (Fig. 2a and  b).In other words, a subpopulation of B. bacteriovorus cells can replicate DNA in PYE10 while others lose viability.The total biomass content and intracellular ATP concentration of the axenically grown B. bacteriovorus also increased up to 2.5-fold and 5-fold, respectively, after 24 h (Fig. 2c and d).Under these growth conditions, we obtained a homogenous population that would allow for a reliable and reproducible analysis of their physiological and metabolic states.Unexpectedly, the cells in the axenic culture were significantly larger than those in the control culture in the HEPES buffer (the mean size of PYE-Bd and HEPES-Bd were 1.27 ± 0.5 and 0.65 ± 0.21 µm, respectively; Mann-Whitnee U-test, p < 0.0001) (Fig. 3), in concordance with previous works measuring cell size under axenic conditions (Dwidar et al. 2017).In contrast, when the predators were inoculated in HEPES buffer, all monitored parameters decreased, indicating cell death.
To confirm the predatory capability of the B. bacteriovorus cells after incubation in prey-free PYE10 rich media, we performed predation experiments on P. putida KT2440.Our analysis of prey viability after 24 h of predation confirms the killing activity of the axenic Bdellovibrio cells, thereby ruling out the possibility of loss of the predatory genotype after cultivation in prey-free conditions (Fig. 3).
The genotype of the wild-type B. bacteriovorus 109J was also verified by sequencing the genes associated with the host-independent (HI) phenotype (bd0108, bd3461, and bd3464 (Cotter and Thomashow 1992)).Sequences were compared with two HI Bdellovibrio mutants, HI18 and HI24 (Prof.Jurkevitch lab collection).No significant mutations were accumulated during the incubation in the absence of prey (Fig. S2).To rule out mutations in other locations, we sequenced the genome of the predator cells after 24 h of axenic incubation in Hepes buffer or PYE10 medium.Comparison of the whole genome sequence of B. bacteriovorus 109J growing in co-culture with prey or axenically did not reveal any significant mutations (Fig. S3 and Supplementary Dataset 2).
Overall, these results demonstrated the active metabolism of the wild-type predator grown in PYE10 media, supported by the active DNA replication, the increased biomass content and intracellular ATP concentration, and the maintenance of the predatory capability.Regarding cell size, our data suggest that incubation in PYE10 results in an increase in cell size (Fig. 3a), even though the 109J strains contain a mutational change in the bd1075 gene related to typical vibrio curvature and shape (Banks et al., 2022).

Analysis of metabolite consumption during Bdellovibrio culture in PYE10 medium
To determine the nutrient composition of the PYE10 medium and monitor the uptake of metabolites by B. bacteriovorus, we conducted a gas-chromatography/quadrupole time-of-flight (GC-QTOF) analysis.Predator cells were incubated in PYE10 medium; after 96 h, the supernatant was collected and analysed.Amino acids constituted 75 % of all nutrients present and detected in the PYE10 medium (Fig. 4a).In addition, the main non-amino acid metabolites detected in the PYE10 medium were compounds related to secondary metabolism such as butanoic, propanedioic, and gluconic acids.The analysis also revealed relatively high amounts of trehalose, xanthine, and pyranose derivatives and phthalic acid after incubation of the predator cells.There were minimal spontaneous variations in the composition of the control medium without predator after 96 h of incubation (Fig. S4 and Supplementary dataset 1), as previously reported (Ames and MacLeod 1985).In contrast, the presence of Bdellovibrio drastically altered the composition of the medium, primarily through the consumption of amino acids (Fig. 4b, Fig. S5, and Supplementary Dataset 1).The relative amounts of glutamate, serine, aspartate, isoleucine, and threonine were significantly lower compared with the control culture at the start of the experiment (unpaired t-test pval < 0.02).The results confirmed the preference consumption of amino acids over the small organic acids detected that were mostly secreted (Fig S4 and Supplementary Dataset 1).

Amino acid-based medium maintains B. bacteriovorus metabolically active
A key limitation of the growth rate of bacteria is nutrient concentration.The above results suggest the use of determined amino acids would be sufficient to support B. bacteriovorus growth.To validate this hypothesis, we designed the CAV medium, which only contains the amino acids detected in the previous GC-QTOF-MS analysis as carbon and nitrogen sources (see the "Materials and Methods" section).To monitor the growth dynamics of B. bacteriovorus in the absence of prey, we incubated the predator cells in the defined CAV medium.The axenic behavior was monitored by measuring the viable cell count, the genome number, the biomass content, and the intracellular ATP concentration.The analysis of these parameters (Fig. 5) revealed the  S1 maintenance of an active metabolism.There was no increase in the viable cell count or genome number under this condition, but the biomass content increased significantly during the 48 h (p < 0.03, Krustal-Wallis t-test).After this point, cells strongly sense starvation, resulting in cell energy depletion and biomass reduction at 96 h.

Discussion
The evolution of biological systems is characterized by their ability to acquire diverse strategies (lifestyles) to adapt to various environments.A significant aspect of this adaptation is the preference for specific carbon and energy sources, which results from metabolic adjustments to the environment.B. bacteriovorus has evolved to thrive in the periplasmic space of the prey cell by using the prey cell's cytoplasmic components (Sockett 2009;Lai et al. 2023).Several reports have contributed valuable insights into the energetic metabolism and growth conditions of Bdellovibrio (Gadkari andStolp 1975, 1976); however, there is still a dearth of evidence regarding the specific nutritional requirements of Bdellovibrio and preferred carbon source as a predator.This study analysed the carbon and energy requirements necessary to support the growth of Bdellovibrio.Understanding these critical factors is vital for potential industrial and medical applications of Bdellovibrio.
The bacterial cytoplasm serves as a highly concentrated compartment, containing a significant portion of the cell's macromolecules (30-40 %) and proteins (over 70 %)  (Zimmerman and Trach 1991).This abundance makes the cytoplasm an exceptionally nutrient-rich environment that facilitates the growth of B. bacteriovorus (Vendeville et al. 2011;Zhang 2011).Most of the cytoplasmic proteins are crowded into the vicinity of the cellular envelope, and prey cellular deformation after the bdelloplast formation likely concentrates and increases accessibility to the protein layer (Baquero et al. 2023).Bdellovibrio proteases can permeate the altered envelope and use these proteins as substrates to obtain small peptides and amino acids.Based on its genome analysis, Bdellovibrio exhibits a wide array of systems dedicated to the uptake of amino acids in the form of di-or tri-peptides (Barabote et al. 2007).
Our research validated the functional metabolism of B. bacteriovorus incubated under axenic conditions predicted by metabolic modelling, specifically using the nutrient-rich medium PYE10, in which the primary ingredients are amino acids.The results of our study confirmed the activation of DNA replication (genome number), ATP generation, and biomass production by Bdellovibrio under these conditions (Fig. 2).Furthermore, intracellular ATP levels increased by 5-fold when the Bdellovibrio cells were incubated in PYE10.The intracellular ATP levels, normalized by biomass, resulted in a 2.5-fold increase, in line with the results obtained in previous studies (Im et al 2018) when incubating B. bacteriovorus strain HD100 cells in NB medium.These differences could be attributed to the different nutrient concentrations in the PYE10 and NB medium since PYE10 provides a much richer, which could allow higher biomass and intracellular ATP concentration values.The increase in these parameters is likely sustained by amino acid consumption, as confirmed by GC-QTOF-MS analysis (Fig. 4).
The predator, Bdellovibrio, is commonly described as host-dependent, which poses a challenge to its efficient application and control.Our findings reveal that Bdellovibrio can be cultured as host-independent bacteria while maintaining its effective killing efficiency (Fig. 3).Even after prolonged starvation conditions without prey, predator cells can reduce the prey population as efficiently as in the Bdellovibrio control culture during the attack phase.This remarkable result highlights the role of Bdellovibrio as a regulator of bacterial populations, not only in aquatic and soil environments (likely invading protein-rich bacterial aggregates or biofilms attached to microbiotic particles (Baquero et al. 2022)) but also in the human commensal microbiota (Iebba et al. 2013;de Dios Caballero et al. 2017).The predator's survival, therefore, does not solely rely on individual predation events.
Furthermore, our experiments pave the way for the rational design of a culture medium to retain the viability of B. bacteriovorus cells and facilitate its use in both medical and biotechnological applications.As demonstrated by Im et al. (2018), the incubation of B. bacterivorus cells in NB medium or S. aureus biofilms resulted in an increased predatory capability and higher intracellular ATP levels as a consequence of amino acid consumption.In this work, we have designed the CAV medium, essentially composed of amino acids that sustain an active metabolism in B. bacteriovorus, leading to an increase in biomass (Fig. 5).Amino acids are precursors of biomass components that fuel the bacterial metabolic network at various points (Wang et al. 2019).For instance, glutamate that enters from α-ketoglutarate plays a key role in anaplerotic reactions.Notably, glutamate dehydrogenase directly fuels the tricarboxylic acid cycle by producing α-ketoglutarate.This reaction serves as an excellent source for reducing equivalents, which are essential metabolites for anabolic reactions.Serine, which enters from 3-phosphoglycerate, supplies the upper path of glycolysis.Leucine and isoleucine enter from pyruvate, connecting with the fatty acid synthesis.This study experimentally validated and confirmed the functional capabilities of the Bdellovibrio proteolytic machinery and the key role of amino acids in sustaining efficient growth, with or without prey.
In summary, the axenic cultivation of B. bacteriovorus under controlled conditions and the modulation of medium composition (PYE10 or CAV) preserves an efficient predator metabolism.Although extensive studies are needed before predator biotechnological and clinical applications become available, the findings presented here open new avenues of research aimed at understanding the nutritional requirements of B. bacteriovorus.

Fig. 1
Fig. 1 Representation of B. bacteriovorus lifecycle and metabolic simulation of its growth capacity.a) During host-dependent growth, B. bacteriovorus cells follow these steps: 1) Prey recognition: Bdellovibrio moves towards prey-rich regions.2) Attachment: Bdellovibrio anchors to the host cell, which leads to the infection.3) Penetration: Bdellovibrio enters the periplasm of the prey cell.4) Growth in bdelloplast and development: the prey appears rounded due to cell wall modification, and Bdellovibrio grows in the periplasm and replicates its DNA.Bdellovibrio uses the prey biomolecules as a source of nutrients.5) Septation: Bdellovibrio septates when resources become

Fig. 2
Fig. 2 Growth parameters of B. bacteriovorus 109J incubated in the absence of prey for 24 h in HEPES buffer and PYE10 rich.a) Viable cell counts of Bdellovibrio measured as pfu/mL.b) Genome number measured using the bd2400 housekeeping gene.c) Measurement of intracellular ATP levels of Bdellovibrio cells.d) Total biomass content.Error bars indicate the standard deviation of three biological replicates.Statistical analyses are listed in TableS1

Fig. 3
Fig. 3 Morphology and predatory ability of B. bacteriovorus after incubations in HEPES or PYE10 medium.Data shown correspond to Bdellovibrio cells incubated in PYE10 for 24 h previously.a) Boxplot representations of the cell size population in µm using ImageJ software.Vertical lines within boxes indicate median values, left and right hinges correspond to the 25th and 75th percentiles, and whiskers extend to observations within 1.5 times the interquartile range (n = 150, Mann-Whitney U test; p-value < 0.01).b) and c) Phasecontrast micrography of Bdellovibrio incubated in HEPES buffer and

Fig. 4
Fig. 4 Relative composition of PYE10 medium during Bdellovibrio axenic incubation.a) Percentage of amino acid and other metabolites of PYE10 medium at the beginning of the experiment and after 96 h of Bdellovibrio incubation.b) Amino acid consumption during Bdellovibrio axenic incubation (Bd 96h ) compared with control culture (PYE10 0h ).Error bars indicate the standard deviation of three bio-